Osteoimmunology is defined as the interplay between the immune system and bone metabolism. This field has developed from the realization that during times of inflammation and stress, immune cells generated in the bone secrete factors that can profoundly affect bone regulation. Leptin is a 16 kDa adipocytokine produced by fat-containing cells. Binding of leptin to central long-form receptors in the hypothalamus stimulates bone loss by decreasing osteoblast function. It has also been demonstrated in vitro that direct binding of leptin to bone marrow stromal cells (BMSCs) can promote their differentiation toward an osteogenic lineage. We hypothesize and have generated preliminary data to suggest that a novel third mechanism of leptin-mediated bone regulation exists. It is hypothesized that leptin acts peripherally in the bone marrow by binding to immune cells such as macrophages to indirectly regulate bone mass through secondary changes in BMSC differentiation and osteoblast function. We will explore these osteoimmunologic interactions to better define the niche of the BMSC and to determine the role our immune system may play in post-natal bone maintenance. To explore our hypothesis in the context of leptin we will employ both in vitro and in vivo techniques. Leptin and leptin-receptor deficient mouse models will be utilized for harvest of primary macrophages and BMSCs. Macrophage cells will be analyzed for their ability to secrete mineralization and differentiation-regulating cytokines through the use of bead-based multichannel ELISA, marketed as BioPlex by Bio-Rad. Differentiation of BMSCs will also be monitored in the presence of control or stimulated macrophages and their products. Finally, local leptin regulation of in vivo bone formation will be explored by re-implanting harvested BMSCs subcutaneously in gelfoam scaffolds. The resulting ossicles will be qualitatively analyzed using micro-CT and immunohistological techniques. This concept of osteoimmunology implies a circle of creation and influence in the marrow cavity and has important implications for the study of inflammation-linked bone disorders including osteoporosis, cancer metastasis to the bone, arthritis, osteomyelitis and periodontal disease.